The long-term goal of Project 4 is to understand how the structure of the skeletal muscle calcium release channel (ryanodine receptor; RyR1) relates to its physical interactions with alpha2s-DHPR and SOCC to form reciprocally regulated Ca 2+release units (CRUs). Equally important cytoplasmic and lumenal interactions with CRU proteins are essential for tight regulation of SR Ca 2+ signaling and will be studied by Project 4 using biochemical, biophysical and cellular approaches to understand the underlying mechanisms relating RyR1 structure to function and its interactions with triadic proteins. Aim I: Define biochemical and biophysical mechanisms underlying functional divergence of wild type (wt)RyR1, wtRyR3, and RyR1/3 chimeras. WtRyR3 and wtRyR1 differ in ability to couple with cqs-DHPR, and exhibit divergent channel gating kinetics, and modulation by physiological and pharmacological ligands. Differential interactions with FKBP12 or 12.6 at the valylprolyl core, as well as long-range allosteric influence may be responsible for these differences. We will define how altered RyR/FKBP interactions relate to divergent phenotypes. Aim II. Elucidate how RyR1 conformation influences coupling with alpha1s,-DHPR and SOCC using point mutants and novel pharmacological agents. The mechanisms by which RyR1 conformation transmits important retrograde information to alpha1s-DHPR and store-operated Ca 2+channels (SOCCs) in myotubes will be defined. Aim III: Define the structural and functional determinants by which Homer proteins regulate the gain of signaling events mediated by RyR1. We will elucidate how Homer proteins physically interact with RyR1 and modulate channel gain. Myotubes from mice lacking expression of one or more Homer protein(s) will be characterized to further define the role of Homer proteins in skeletal e-c coupling. Aim IV. Define how deletion of CaM-binding domains of RyR1 (AA3614-3643) and CaM binding mutations of alpha1s-DHPR expressed in myotubes, alter their functional and physical interactions, and the influence of CaM on these interactions. Determinants for interactions between full-length RyR1 and (alpha1s-DHPR will be elucidated in dvsgenic and dvspedic myotubes.